Fasting condition as dietary treatment of diabetes

ABSTRACT

A method of alleviating symptoms of, or treating, pancreatic beta-cell damage in a subject includes a step of identifying a subject having pancreatic beta-cell damage. Multiple cycles of a diet protocol are administered to the subject. The diet protocol includes administering of a fasting mimicking diet and a re-feeding diet where the fasting mimicking diet is provided for a first time period and the re-feeding diet is provided for a second time period.

CROSS REFERENCE TO RELATED APPLICATION

This application is a continuation of U.S. Ser. No. 14/320,996 filedJul. 1, 2014, now U.S. Pat. No. 9,386,790 issued Jul. 12, 2016, whichclaims the benefit of U.S. provisional application Ser. No. 61/841,709filed Jul. 1, 2013, the disclosures of which are incorporated in theirentireties by reference herein.

STATEMENT REGARDING FEDERALLY SPONSORED RESEARCH OR DEVELOPMENT

The invention was made with Government support under Contract Nos.PO1AG034906 and PO1AG020642. The Government has certain rights to theinvention.

TECHNICAL FIELD

The present invention, in general, relates to compositions and methodsfor a) regenerating pancreatic cells, 2) treating Type I diabetes, 3)treating Type II diabetes, 4) treating the metabolic syndrome, 5)preventing Type II diabetes and other diseases associated with metabolicsyndrome. In particular, the present invention promotes pancreaticregeneration and alleviates multiple common signs, symptoms and riskfactors of both diabetes types. In addition to promoting residentialpancreatic regeneration, it can also be used to enhance and improve theregenerative functions of transplanted stem cells when it isincorporated with the conventional stem cell therapy.

BACKGROUND

Human metabolic disorders such as diabetes mellitus Type I, diabetesmellitus Type II, metabolic syndrome, and insulin resistance are serioushealth conditions affecting over a third of the adult population in theUnited States. Although effective in delaying morbidity, standardtreatments have not generally been able to reverse the associated damageof these disorders.

Chemicals, cytokines/hormones and stem cell or islet transplantationthat boost or assist pancreatic regeneration have been used as diabetestherapy in order to augment or replace insulin injections by increasingthe number of, or enhancing the function of, endogenousinsulin-producing β-cells.

Diabetes mellitus Type 1 and Type 2 diabetes are characterized byprogressive beta-cell failure. By far, stem/progenitor celltransplantation is the only therapy available for advanced stages ofdiabetes in an attempt to restore insulin production and replace insulininjection. However, besides ethical issues, technical and safetychallenges in stem cell isolation, maintenance, expansion,donor-recipient matching and transplantation limit the efficacy of thesestrategies.

Accordingly, there is a need for safe, effective treatment protocols formetabolic disorders such as diabetes that may reverse pancreatic damageand inhibit development of these disorders.

SUMMARY

In at least one embodiment, the present invention provides a method ofalleviating symptoms of or treating pancreatic beta-cell damage in asubject. The method includes a step of identifying a subject havingpancreatic beta-cell damage. Multiple cycles of a diet protocol areadministered to the subject. The diet protocol includes administering ofa fasting mimicking diet and a re-feeding diet where the fastingmimicking diet is provided for a first time period and the re-feedingdiet is provided for a second time period.

In another embodiment, a method of alleviating symptoms of or treatingpancreatic beta-cell damage in a subject is provided. The methodincludes a step of identifying a subject having pancreatic beta-celldamage and insulin deficiency. The subject's normal caloric intake isdetermined. Multiple cycles of a diet protocol are administered to thesubject. The diet protocol includes administering of a fasting mimickingdiet and a re-feeding diet where the fasting mimicking diet is providedfor a first time period (e.g., 2 to 6 days) and the re-feeding diet isprovided for a second time period (e.g., 7 to 85 days). The fastingmimicking diet provides less than about 50% of the normal caloric intakeof the subject with both protein restriction and sugar restriction andthe re-feeding provides 60-100 percent of the normal caloric intake ofthe subject, depending on the need to lose additional weight.

In another embodiment, a method of alleviating a symptom of diabetes isprovided. The method includes a step of identifying a subject havingdiabetes (Type I or II). The subject is provided with multiple cycles ofa Fasting Mimicking Diet (FMD) (4-5 days every, 1-12 weeks) to promote areduction and reversal in symptoms.

In another embodiment, a method of alleviating insulin resistance,insulin deficiency and/or hyperglygemia is provided. The method includesa step of identifying a subject having insulin resistance, insulindeficiency and/or fasting hyperglycemia diabetes. The subject isprovided with multiple cycles of a Fasting Mimicking Diet (FMD) (e.g.,4-5 days every 4-12 weeks) to promote a reduction in symptoms.

In another embodiment, a method of alleviating a symptom of metabolicsyndrome is provided. The method includes a step of identifying asubject having one or more metabolic syndrome symptoms/risk factors(e.g., high blood pressure, hyperglycemia, excess body fat around thewaist, high cholesterol). The subject is provided with multiple cyclesof a Fasting Mimicking Diet (FMD) (4-5 days every, 1-12 weeks) topromote a reduction in symptoms.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1. Table 1 showing the Calorie overview of the fasting mimickingdiet adjusted to human subjects. The fasting mimicking diet (FMD),Prolon, induces a fasting-like response while maximizing nourishment.The consumed calories for each one of the 5 days of the diet are shown,as well as the adjusted kcal per pound and kilogram of body weight. Thereduction in calories consumed during the 5 day dietary regimen (Δ5-day)is shown as either 1) based on a 2,000 calorie per day diet, or 2) basedon 2,800, 2,400, and 2,000 calorie diets for person's weight ≥200,150-200, and ≤150 lbs, respectively;

FIG. 2. Table 2 showing the defined macronutrient content for each dietday adjusted to a 180-200 lbs human subject. The macronutrient contentfor each day of the 5 day FMD regimen is based on an average 180-200 lbsperson. Caloric intake on day 1 of the diet is less reduced compared tothe following days (2-5) to allow the body to adjust to the low calorieconsumption. % of calories contributed by fat, carbohydrate (by sugar indetail) and protein for each day of the Prolon regimen is presented.

FIG. 3. Table 3 showing the defined micronutrient content for each dietday adjusted to a 180-200 lbs human subject in a variation of theinvention. The micronutrient content for each day of the 5 day FMDregimen based on an average 180-200 lbs person. Percent of the dailyvalue (% DV) is calculated based on a 2,000 calorie diet. * for some ofthe micronutrients, DV is not defined; values shown are based on thereference daily intake (RDI).

FIGS. 4A, 4B, 4C, 4D, and 4E. Fasting mimicking diet (FMD, as describedin FMD1 and FMD2 sections) in Type I diabetes mice. (A) Blood glucoselevels of mice with STZ-induced hyperglycemia. Vertical dash linesindicate the cycles of FMD. Horizontal dash line indicates the level ofblood glucose in healthy controls. (B) Plasma insulin levels ofSTZ-treated mice with or without FMD. Horizontal dash line indicates thelevel of plasma insulin in healthy controls (1.81±0.25 ug/L). (C)Glucose tolerance and Insulin tolerance test. Mice were injected withglucose or insulin and the blood glucose levels were measured over a 60min period. (D) Immunofluorescence staining of pancreatic islets forinsulin (bright area). In FMD group, both rescued (euglycemic) andunrescued (pre-hyperglycemic) mice contained more insulin-secretingbeta-cells comparing to the AL group. (E) Survival curve of STZ treatedmice.

FIGS. 5A and 5B. Fasting mimicking diet (FMD as described in FMD1 andFMD2 sections) in Type II diabetes mice (lepr^(db/db)). (A) Body weightand (B) blood glucose level of db/db mice. 8-wks-old db/db mice were fedad libitum or FMD. Vertical dash lines indicate the cycles of FMD.

FIG. 6. Effect of Fasting and a Fasting Mimicking Diet (as described inFMD1 and FMD2 sections) in a pre-diabetic human subject. Fasting plasmaglucose (FPG) levels in a pre-diabetic subject who underwent multiplecycles of a FMD. Reduction in blood glucose was found after cycles offasting/diet cycles, at both the non-fasting phase and fasting/dietphase. Horizontal line indicates the level for identifying pre-diabetes(FPG 110-125 mg/dl).

FIG. 7. An experimental scheme of a human adapted version of the FastingMimicking Diet (FMD).

FIG. 8 provides the distribution of fasting blood glucose of humansubjects before (A) and after (C) receiving FMD.

FIG. 9. Circulating levels of betatrophin, shown to cause pancreaticbeta cell regeneration, were increased at post-FMD refeeding.

FIG. 10. FMD cycles showing a reduction in cytokines associated withautoimmune type 1 diabetes in mice.

DETAILED DESCRIPTION

As required, detailed embodiments of the present invention are disclosedherein; however, it is to be understood that the disclosed embodimentsare merely exemplary of the invention that may be embodied in variousand alternative forms. The figures are not necessarily to scale; somefeatures may be exaggerated or minimized to show details of particularcomponents. Therefore, specific structural and functional detailsdisclosed herein are not to be interpreted as limiting, but merely as arepresentative basis for teaching one skilled in the art to variouslyemploy the present invention.

The terms “kilocalorie” (kcal) and “Calorie” refer to the food calorie.The term “calorie” refers to the so-called small calorie.

The term “subject” refers to a human or animal, including all mammalssuch as primates (particularly higher primates), sheep, dog, rodents(e.g., mouse or rat), guinea pig, goat, pig, cat, rabbit, and cow.

The term “metabolic syndrome” as used herein means a disorder thatincreases a subject's risk of cardiovascular disease, stroke, and TypeII diabetes. The syndrome includes the following metabolic risksfactors: a large waistline, high blood pressure, low high densitylipoprotein (HDL) levels (<40 mg/dL), and high fasting glucose levels(or a subject on medication to treat high glucose levels). Typically, asubject having three or more of these risk factors is classified ashaving metabolic syndrome.

The term “fasting glucose level” means the glucose level in the blood ofa subject after 8 hours of fasting. In human subjects, the normalfasting glucose blood level is less than 100 mg/dL (e.g. 70 to 99mg/dL).

Abbreviations:

“STZ” is Streptozotocin.

“FMD” is fasting mimicking diet.

“AL” is ad libitum.

In an embodiment, a method of alleviating symptoms of or treatingpancreatic beta-cell damage in a subject is provided. In this context,pancreatic beta-cell damage includes beta cell loss, beta celldysfunction, or combinations thereof In variations of the presentembodiment, the method is used to treat or alleviate one or moresymptoms of diabetes mellitus Type I, diabetes mellitus Type II,metabolic syndrome, and insulin resistance. The method includes a stepof identifying a subject having pancreatic beta-cell damage. Thesubject's normal caloric intake is typically determined by interrogationor from the subject's weight. Multiple cycles of a diet protocol areadministered to the subject. The diet protocol includes administering ofa fasting mimicking diet and a re-feeding diet where the fastingmimicking diet is provided for a first time period and the re-feedingdiet is provided for a second time period.

The fasting mimicking diet provides the subject with less than about 50%of the normal caloric intake of the subject. In a variation the fastingmimicking diet is provided with protein restriction and/or carbohydraterestriction and/or sugar restriction. In one useful refinement, there-feeding provides 60-100 percent of the normal caloric intake of thesubject. In a refinement, the fasting mimicking diet provides thesubject with at most, in increasing order of preference, 60%, 50%, 45%,40%, or 35% of the normal caloric intake of the subject with bothprotein restriction and carbohydrate/sugar restriction and at least, inincreasing order of preference, 0%, 5%, 10%, or 15% of the normalcaloric intake of the subject with both protein restriction and sugarrestriction. The re-feeding provides the subject with at least 60percent of the normal caloric intake of the subject. In a refinement,the re-feeding diet provides the subject with at least, in order ofpreference, 60%, 70%, 80%, 90%, or 100% of the normal caloric intake ofthe subject and at most, in order of preference, 130%, 120%, 110%, or105% of the normal caloric intake of the subject. In a variation, there-feeding diet provides an amount of calories that depends on the needof the subject to lose weight. For example, a subject needing weightreduction can be provided a re-feeding diet that provides from 60 to 90percent of their normal caloric intake. In a refinement, a subjectneeding weight reduction can be provided a re-feeding diet that providesfrom 60 to 80 percent of their normal caloric intake. In anotherrefinement, a subject needing weight reduction can be provided are-feeding diet that provides from 60 to 70 percent of their normalcaloric intake. Subject not requiring weight loss can be provided withfrom 90 to 130 percent of their normal caloric intake during there-feeding diet phase.

In a variation, the fasting mimicking diet provides the subject with 4.5to 7 kcal/pound of body weight/day on day 1 followed by 3 to 5 kcalkcal/pound of body weight/day for days 2 to 5. In a refinement, thefasting mimicking diet provides the subject with 7 kcal/pound of bodyweight/day on day 1 followed by 4 kcal kcal/pound of body weight/day fordays 2-5. In another variation, the fasting mimicking diet provides thesubject with 3-6 kcal/pound of body weight/day on day 1 followed by 2-4kcal kcal/pound of body weight/day for days 2-4. In some variations, thefasting mimicking diet includes 2 to 5% calories from glycerol. Forexample, the fasting mimicking diet can include at least 60% caloriesfrom fatty acids, 2-5% calories from glycerol and up to 5% of caloriesfrom plant-based proteins, and a maximum of 35% of calories fromcarbohydrates. Advantageously, the carbohydrates are complexcarbohydrate from plant sources such as soy, rice, or other grains andat least 50% of the calories from fatty acids are from coconut oil andtree nuts (e.g., macadamia nuts, walnuts, or almonds).

Typical fat sources include vegetable oil such as soybean oil. In afurther refinement, the low protein diet includes fat sources such thatat least 25 percent of calories from fat are short-chain fatty acidshaving from 2 to 7 carbon atoms and/or from medium-chain saturated fattyacids having from 8 to 12 carbon atoms. Specific examples of fatty acidsinclude lauric and/or myristic acid and fat sources include olive oil,kernel oil and/or coconut oil. In another refinement, the fastingmimicking diet includes calories from fat in an amount from about 0 to22 percent of total calories contained in the diet.

U.S. patent application Ser. No. 14/178,953 filed on Feb. 12, 2014provides examples of fasting mimicking diets that are useful in themethods of the present invention. In this regard, FIGS. 1-3 providelistings of the nutrients for day one through day five. In addition tothe macronutrients, the diet should contain less than 30 g of sugar onday 1 and less than 20 g of sugar on days 2-5. The diet should containless than 28 g of proteins on day 1 and less than 18 g of proteins ondays 2-5, mostly or completely from plant based sources. The diet shouldcontain between 20 and 30 grams of monounsaturated fats on day 1 and10-15 grams of monounsaturated fats on days 2-5. The diet should containbetween 6 and 10 grams of polyunsaturated fats on day 1 and 3-5 grams ofpolyunsaturated fats on days 2-5. The diet should contain less than 12 gof saturated fats on day 1 and less than 6 grams of saturated fats ondays 2-5. Typically, the fats on all days are derived from a combinationof the following: Almonds, Macadamia Nuts, Pecans, Coconut, Coconut oil,Olive Oil and Flaxseed. In a refinement, the FMD diet includes over 50%of the recommended daily value of dietary fiber on all days. In thefurther refinement, the amount of dietary fiber is greater than 15 gramsper day on all five days. The diet should contain 12-25 grams ofglycerol per day on days 2-5. In a refinement, glycerol is provided at0.1 grams per pound body weight/day. In a variation, the FMD includesthe following micronutrients (at least 95% non-animal based): over 5,000IU of vitamin A per day (days 1-5); 60-240 mg of vitamin C per day (days1-5); 400-800 mg of Calcium per day (days 1-5); 7.2-14.4 mg of Iron perday (days 1-5); 200-400 mg of Magnesium per day (days 1-5); 1-2 mg ofcopper per day (days 1-5); 1-2 mg of Manganese per day (days 1-5); 3.5-7mcg of Selenium per day (days 1-5); 2-4 mg of Vitamin B1 per day (days1-5); 2-4 mg of Vitamin B2 per day (days 1-5); 20-30 mg of Vitamin B3per day (days 1-5); 1-1.5 mg of Vitamin B5 per day (days 1-5); 2-4 mg ofVitamin B6 per day (days 1-5); 240-480 mcg of Vitamin B9 per day (days1-5); 600-1000 IU of Vitamin D per day (days 1-5); 14-30 mg of Vitamin Eper day (days 1-5); over 80 mcg of Vitamin K per day (days 1-5); 16-25mcg Vitamin B12 are provided during the entire 5-day period; 600 mg ofDocosahexaenoic acid (DHA, algae-derived) are provided during the entire5-day period. The FMD diet provides high micronutrient content mostly(i.e., greater than 50 percent by weight) from natural sourcesincluding: Kale, Cashews, Yellow Bell Pepper, Onion, Lemon Juice, Yeast,Turmeric. Mushroom, Carrot, Olive Oil, Beet Juice, Spinach, Tomato,Collard, Nettle, Thyme, Salt, Pepper, Vitamin B12 (Cyanocobalamin),Beets, Butternut Squash, Oregano, Tomato Juice, Orange Juice, Celery,Romaine Lettuce, Cumin, Orange Rind, Citric Acid, Nutmeg, Cloves, andcombinations thereof. Table 4 provides an example of additionalmicronutrient supplementation that can be provided in the FMD diet:

TABLE 4 Micronutrient Supplementation Supplement Formula Amount AmountRange Unit Vit A 1250 IU  900-1600 IU Vit C Ascorbic Acid C₆H₈O₆ 15.000010-20 mg Ca Calcium CaCO₃ 80.0000  60-100 mg Carbonate Fe FerrousFumarate C₄H₂FeO₄ 4.5000 3-6 mg Vit D3 Cholecalciferol C₂₇H₄₄O 0.00250.001-0.005 mg Vit E dl-Alpha C₂₉H₅₀O₂ 5.0000 3-7 mg Tocopheryl AcetateVit K Phytonadione 0.0200  0.1-0.04 mg Vit B1 Thiamine C₁₂H₁₇N₅O₄S0.3750 0.15-0.5  mg Mononitrate Vit B2 Riboflavin E101 C₁₇H₂₀N₄O₆ 0.42500.2-0.6 mg Vit B3 Niacinamide C₆H₆N₂O 5.0000 3-7 mg Vit B5 CalciumC₁₈H₃₂CaN₂O₁₀ 2.5000 1.5-4.0 mg Pantothenate Vit B6 PyridoxineC₈H₁₁NO₃•HCl 0.5000 0.3-0.7 mg Hydrochloride Vit B7 Biotin C₁₀H₁₆N₂O₃S0.0150 0.01-0.02 mg Vit B9 Folic Acid C₁₉H₁₉N₇O₆ 0.1000 0.07-0.14 mg VitB12 Cyanocobalamin C₆₃H₈₈CoN₁₄O₁₄P 0.0015 0.001-0.002 mg Cr ChromiumCr(C6H4NO2)3 0.0174 0.014-0.022 mg Picolinate Cu Cupric Sulfate CuSO40.2500 0.18-0.32 mg I Potassium Iodide KI 0.0375  0.03-0.045 mg MgMagnesium MgO 26.0000 20-32 mg Oxide Mn Manganese MnSO₄ 0.5000 0.3-0.7mg Sulfate Mo Sodium Na₂MoO₄ 0.0188 0.014-0.023 mg Molybdate Se SodiumSelenate Na₂O₄Se 0.0175 0.014-0.023 mg Zn Zinc Oxide ZnO 3.7500 3-5 mg

In a variation as set forth above, the fasting mimicking diet alsoincludes caloric restriction with respect to proteins and sugars (e.g.,glucose). In one refinement, the fasting mimicking diet includes proteinin an amount that is less than 15 percent of total calories provided bythe fasting mimicking diet. In a further refinement, the fastingmimicking diet includes protein in an amount that is at most, inincreasing order of preference, 15%, 12%, 10%, 8%, or 5% of totalcalories provided by the fasting mimicking diet and in an amount that isat least 0%, 2%, 3%, 5%, or 6% of total calories provided by the fastingmimicking diet. In another refinement, the fasting mimicking dietincludes sugars in an amount that is less than 15 percent of totalcalories provided by the fasting mimicking diet. In a furtherrefinement, the fasting mimicking diet includes sugars (e.g., glucose)in an amount that is at most, in increasing order of preference, 15%,12%, 10%, 8%, or 5% of total calories provided by the fasting mimickingdiet and in an amount that is at least, in order of preference, 0%, 2%,3%, 5%, or 6% of total calories provided by the fasting mimicking diet.

The first time period during which the fasting mimicking diet isprovided is typically from 2 to 6 days and the second time period duringwhich the re-feeding diet is provide is typically 7 to 85 days. In avariation, the multiple cycles are administered once a month for atleast 3 months. In a refinement, the first time period during which thefasting mimicking diet is provided for is, in increasing order ofpreference, 2, 7, 3, 6, 4, or 5 days. In another refinement, the secondtime period during which the re-feeding diet is provided is from, inincreasing order of preference, 1 to 6 weeks, 1 to 5 weeks, 1 to 4weeks, 1 to 3 weeks, or 1 to 2 weeks. The multiple cycles of the dietprotocol may continue indefinitely. Advantageously, the multiple cyclesof the diet protocol lasts for at least 60 days. In a refinement, themultiple cycles of the diet protocol lasts for at least, in order ofpreference, 30 days, 60 days, 90 days, 120 days, 180 days, 270 days, 1year, 2 years, 5 years, or for the lifetime of the subject.

Surprisingly, the method and FMD of the present embodiment is found toinduce pancreas and beta cell regeneration. This aspect of the inventionis useful in treating subjects exhibiting both insulin resistance andthe need for new beta cells (e.g., late stage type 2 diabetes) andsubjects needing beta cell regeneration but do not have insulinresistance (e.g., type 1 diabetics). In another aspect, the method andFMD is found to increase the insulin sensitizer adiponectin, promote theloss of abdominal fat and cause insulin sensitization and a decrease infasting glucose levels. Advantageously, the ability of the diet toreverse insulin resistance is useful in preventing the development ofdiabetes Type II in subjects exhibiting insulin resistance and/orfasting hyperglycemia but not having a diagnosis of diabetes. However,the diet is also useful for a normal subject who may be at risk ofdeveloping insulin resistance, metabolic syndrome or diabetes for anyreason including excess adiposity, poor diet, genetic risk factors fordiabetes etc.

Cycles of the fasting FMD a) promote insulin producing β-cellsregeneration, b) prevents and reverses severe hyperglycemia (≥300 mg/dl)in both Type I and Type II diabetes in mammals (mice) and c) alleviatesother complications of diabetes including insulin secretion deficiency,glucose tolerance impairment and insulin insensitivity. It alsodecreases fasting blood glucose levels in a pre-diabetic human subject,and it increases the insulin sensitizer adiponectin and the pancreaticregeneration factor betatrophin in humans confirming that this efficacyis conserved in mice and humans. This high efficiency diet protocol hasbroad effects on multiple complications of diabetes, low initialrequirements and long-term safety/benefits allowing this invention to bepractically incorporated with various types of therapy, including otherconventional regenerative approaches as well as standard treatments ofdiabetes (under physician supervision) in a way that directly stimulatesthe resident stem cells and/or may indirectly change themicroenvironments for promoting the regeneration of the transplantedstem cells in the recipients. The pancreatic regeneration caused by theFMD is only part of its effects on Types 1 and 2 diabetes. In fact, theFMD causes a reduction in glucose, reduction in IGF-I, decrease ininsulin and an increase in insulin sensitivity, all of which areindependent or partly independent of regeneration and contribute to theanti-diabetes effects. In a refinement, the subject's levels of glucose,IGF-I, insulin and insulin sensitivity are measured to verify thecorrect changes.

In another embodiment, a method of alleviating a symptom of diabetes isprovided. The method includes a step of identifying a subject sufferingfrom diabetes (Types I or II). The subject is provided with multiplecycles of a Fasting Mimicking Diet (FMD) (4-5 days every, 1-12 weeksdepending on severity of symptoms and levels of risk factors) to promotea reduction in symptoms. Cycles of the fasting FMD a) promote insulinproducing (3-cells regeneration, b) prevents and reverses severehyperglycemia (≥300 mg/dl) in both Type I and Type II diabetes inmammals (mice) and c) alleviates other complications of diabetesincluding insulin secretion deficiency, glucose tolerance impairment andinsulin insensitivity. It also decreases fasting blood glucose levels ina pre-diabetic human subject, confirming that this efficacy is conservedin mice and humans. It also decreases abdominal fat and increases thelevels of the insulin sensitizer adiponectin. This high efficiency dietprotocol has broad effects on multiple complications of diabetes, lowinitial requirements and long-term safety/benefits allowing thisinvention to be practically incorporated with various types of therapy,including other conventional regenerative approaches as well as standardtreatments of diabetes (under physician supervision) in a way thatdirectly stimulates the resident stem cells and/or may indirectly changethe microenvironments for promoting the regeneration of the transplantedstem cells in the recipients. The pancreatic regeneration caused by theFMD is only part of its effects on Types 1 and 2 diabetes. In fact, theFMD causes a reduction in glucose, reduction in IGF-I, decrease ininsulin and an increase in insulin sensitivity, all of which areindependent or partly independent of regeneration and contribute to theanti-diabetes effects. In a refinement, the subject's levels of glucose,IGF-I, insulin and insulin sensitivity are measured to verify thecorrect changes.

Treatment and Prevention of Type 1 Diabetes.

Subjects with pancreatic beta-cell damage leading to partial or fullType I diabetes undergo multiple cycles of the FMD 1 or 2 diets asdescribed herein. The number of cycles will depend on the severity ofthe disease and on the effect of each cycle of the FMD on beta cellregeneration and insulin production. The FMD will substitute a subject'snormal diet for a period of 4-5 days every 1-4 weeks depending on thetype and severity of the diabetes and insulin resistance, with the morefrequent FMD prescribed to subjects with a more severe form of Type 1diabetes. The frequency of the diet will also depend on the ability ofsubjects to return to within 5% of the normal weight before starting thenext cycle of the diet and on the amelioration of the diabetes symptoms,with discontinuation or major reduction of the cycles once the values ofinsulin and glucose return to the normal range. For morbidly obesesubjects, the FMD could be applied every 2 weeks for 5 days. The dietconsists of ingredients which are Generally Regarded As Safe (GRAS) (seebelow). Because of the combination of potent immunomodulatory andanti-inflammatory effects, the FMD regimen described above can also beused to prevent the progression of Type 1 diabetes.

Treatment of Type 2 Diabetes.

Subjects with insulin resistance and/or pancreatic beta-cell damageleading to partial of full Type II diabetes will be asked to undergomultiple cycles of the FMD diet as described herein. The number andfrequency of cycles can be determined by the physician depending on theseverity of the disease and on the effect of each cycle of the FMD oninsulin resistance, glucose tolerance, beta cell regeneration andinsulin production and on the ability of the subject to maintain theseimprovements. Depending on the stage of the diabetes, insulin productionmay not be affected. The FMD will substitute a subject's normal diet fora period of 4-5 days every 1-12 weeks depending on the type and severityof the diabetes and insulin resistance, with the more frequent FMDprescribed to subjects with a more severe form of Type 2 diabetes,higher insulin resistance and body mass index. For subject within anormal BMI (<25), the frequency of the diet will also depend on theability of subjects to return to within 5% of the normal weight beforestarting the next cycle of the diet. For overweight and obese subjects,the FMD could be applied as frequently as every week, based on theassessment of the physician. The diet consists of ingredients, which areGenerally Regarded As Safe (GRAS) (see below).

FMD 1 (5 days): a low protein and low carbohydrate diet containing no orminimal animal derived components. The diet provides 7 kcal/pound ofbody weight for day 1 followed by 4 kcal/pound of body weight/day fordays 2-5. At least 60% of calories are from a composition of fattyacids, preferably 100% from plant sources (with 50% or more coming fromcoconut oil and nuts including macadamia, walnuts, almonds), glycerol(2-5% of kcal) and 5% of calories from plant-based proteins (soy, rice,other grains)+a maximum of 35% of calories from carbohydrates mostlycomplex from plant sources. The diet is also high nourishment andprovides, on each day, 30-50% of the daily recommended intake for allvitamins and minerals+essential fatty acids, with at least 50% of themcoming from natural sources.

FMD 2 (4 days): a low protein and low carbohydrate diet containing no orminimal animal derived components. The diet provides 3-5 kcal/pound ofbody weight for day 1 followed by 2-4 kcal/pound of body weight/day fordays 2-4. At least 60% of calories are from a composition of fatty acids(with 50% or more coming from coconut oil and nuts including macadamia,walnuts, almonds), glycerol (2-5% of kcal) and 5% of calories fromplant-based proteins (soy, rice, other grains)+a maximum of 35% ofcalories from carbohydrates, mostly complex, from plant sources. Thediet is also high nourishment and provides, on each day, 30-50% of thedaily recommended intake for all vitamins and minerals+essential fattyacids, with at least 50% of them coming from natural sources.

In another embodiment, a method of alleviating or preventing insulinresistance and/or hyperglycemia is provided. The method includes a stepof identifying a subject having insulin resistance and/or fastinghyperglycemia diabetes. In this regard, subjects having a family historyof, or showing trending towards, insulin resistance and/or fastinghyperglycemia diabetes can also be treated to inhibit the development ofthese conditions. Indications of a subject trending towards insulinresistance and/or hyperglygemia include increasing weight, increasingfasting glucose levels, increasing hyperglycemia, increasing insulinresistance, and the like over several months to years (e.g., 6 months to5 years). The subject is provided with multiple cycles of the fastingmimicking diet as set forth above for treating beta-cell damage anddiabetes. In one particular variation, the FMD is provided to suchsubjects for 4-5 days every 4-12 weeks to promote a reduction insymptoms.

In another embodiment, a method of alleviating a symptom of orpreventing metabolic syndrome is provided. The method includes a step ofidentifying a subject having one or more metabolic syndromesymptoms/risk factors. Examples of such symptoms/risk factors includehigh blood pressure, hyperglycemia, excess body fat around the waist,high total cholesterol, low HDL, and the like. In this regard, subjectshaving a family history of or showing trending towards metabolicsyndrome can also be treated to inhibit the development of theseconditions. Indications of a subject trending towards metabolic syndromeinclude a decreasing HDL, increasing total cholesterol, increasingweight, increasing fasting glucose levels, increasing hyperglycemia,increasing insulin resistance, and the like over several months to years(e.g., 6 months to 5 years). The subject is provided with multiplecycles of a Fasting Mimicking Diet (FMD) as set forth above for thetreatment of beta-cell damage and diabetes. In one particular variation,the subject is provided with the FMD 4-5 days every 1-12 weeks topromote a reduction in symptoms.

In another embodiment, a diet package for implementing the fastingmimicking diets set forth above is provided. In general, the dietpackage includes rations for implementing the fasting mimicking dietsset forth above. The sources of these rations are also set forth above.In particular, the rations are divided in portions for each cycle of thefasting mimicking diet. Alternatively, the rations are divided intoportions for each day of the fasting mimicking diet. In anothervariation, the rations are divided into portions for each meal of thefasting mimicking diet. For example, the diet package includes a firstset of rations for a fasting mimicking diet to be administered for thefirst time period to a subject, the fasting mimicking diet providingfrom 4.5 to 7 kilocalories per pound of subject for a first day and 3 to5 kilocalories per pound of subject per day for a second to fifth day ofthe low protein diet. The diet package includes rations that provideless than 30 g of sugar on the first day; less than 20 g of sugar on thesecond to fifth days; less than 28 g of proteins on the first day; lessthan 18 g of proteins on the second to fifth days; 20 to 30 grams ofmonounsaturated fats on the first day; 10 to 15 grams of monounsaturatedfats on the second to fifth days; between 6 and 10 grams ofpolyunsaturated fats on the first day; 3 to 5 grams of polyunsaturatedfats on the second to fifth days; less than 12 g of saturated fats onthe first day; less than 6 grams of saturated fats on the second tofifth days; and 12 to 25 grams of glycerol per day on the second tofifth days. In a refinement, the diet package further includessufficient rations to provide the micronutrients set forth above. In afurther refinement, the diet package provides instructions providingdetails of the methods set forth above.

The substitution diets are scaled for mice in the experiments set forthbelow to obtain equivalent changes in IGF-I (over 30% reduction), IGFBP1(over 5 fold increase), ketone bodies (detection of high levels of Bhydroxyl butyrate), glucose (at least 20% reduction) and insulin (atleast 20% reduction). The FMD can be given as cycles of a preventive ortherapeutic diet, every 1-12 weeks (see above) followed by a normaldiet. Subsequent cycles must be postponed until the subject returns to aBMI of at least 18 or what is considered by the physician to be ahealthy weight.

The following examples illustrate the various embodiments of the presentinvention. Those skilled in the art will recognize many variations thatare within the spirit of the present invention and scope of the claims.

Mouse FMD Experiments

FIGS. 4A-E provide the results of the fasting mimicking diet (FMD) inType I diabetes mice as described in FMD1 and FMD2 sections set forthabove. FIG. 4A provides blood glucose levels of mice with STZ-inducedhyperglycemia. The vertical dash lines indicate the cycles of FMD whilethe horizontal dash line indicates the level of blood glucose in healthycontrols. FIG. 4B provides plasma insulin levels of STZ-treated micewith or without FMD. FIG. 4C provides glucose tolerance and insulintolerance test results. Mice were injected with glucose or insulin andthe blood glucose levels were measured over a 60 min period. FIG. 4Dprovides immunostaining of pancreatic islets for insulin (bright area).In the FMD group, both rescued and unrescued mice contained moreinsulin-secreting beta-cells comparing to the AL group. Finally, FIG. 4Eprovides a survival curve of STZ treated mice.

FIGS. 5A-B provide the results of the fasting mimicking diet in Type IIdiabetes mice (lepr^(db/db)) (as described in FMD1 and FMD2 sections).FIG. 5A provides the body weight while FIG. 5B provides the bloodglucose level of db/db mice. In these experiments, 8-wks-old db/db micewere fed ad libitum or FMD. Vertical dash lines indicate the cycles ofFMD, while the horizontal dash line indicates the blood glucose levelsof non-diabetic controls.

FIG. 6 provides the effect of Fasting and a Fasting Mimicking Diet (asdescribed in FMD1 and FMD2 sections) in a pre-diabetic human subject.With respect to fasting plasma glucose (FPG) levels in a pre-diabeticsubject who underwent multiple cycles of a FMD, reduction in bloodglucose was found after cycles of fasting/diet cycles, at both thenon-fasting phase and fasting/diet phase. The horizontal line indicatesthe level for identifying pre-diabetes (FPG 110-125 mg/dl).

Human FMD Trials

FIG. 7 illustrates an experimental scheme of a human adapted version ofthe Fasting Mimicking Diet (FMD). FMD was supplied to participants onceper month for 3 consecutive cycles. Each cycle consists of 5 days of FMDand 25 days of refeeding. Biomedical measurements presented below wereperformed at baseline (E) and prior to the start of the diet (A),immediately after the first FMD cycle (B) and during the refeedingperiod after the 1st cycle (C) and that after 3 cycles of FMD+Refeeding(D).

FIG. 8 provides the distribution of fasting blood glucose of humansubjects. FMD reduces blood glucose levels in human subjects originallywith high basal blood glucose (baseline >90 mg/dl). Fasting bloodglucose was measured at indicated time points: A, before receiving FMD;C, 7 days of refeeding after 1st FMD. See also FIG. 7 for theexperimental scheme. N=16.

Table 5 provides the levels of fasting blood glucose of human subjectsbefore and after receiving FMD. FMD reduces blood glucose levels inhuman subjects with high basal blood glucose (>90 mg/dl) (p<0.05,t-test). Fasting blood glucose was measured at indicated time points:pre-FMD, tme point A, before receiving FMD; post-FMD, time point C, 7days of refeeding after 1st FMD. See also FIG. 7 for the experimentalscheme.

TABLE 5 Levels of fasting blood glucose of human subjects before andafter receving FMD pre-FMD post-FMD (n = 8) (n = 8) Minimum 93.00 87.0025% Percentile 94.63 93.00 Median 98.25 95.00 75% Percentile 102.5 97.50Maximum 109.5 101.0

Tables 6A and 6B gives the homeostasis model assessment of insulinresistance (HOMA-IR) and beta cell function (% B) of human subjects onFMD. HOMA-IR and % B at indicated time points are used here to evaluatethe contribution of insulin sensitivity or beta cell function to theeffects of FMD on steady-state glucose homeostasis. Indexes werecalculated using fasting blood glucose and plasma insulin levels atindicated time points: E, baseline; A, before FMD; B, at the end of 1stFMD; C, refeeding period after 1st FMD; D refeeding period after 3cycles of FMD. Trends of induction or reduction were indicated by thearrows. See also FIG. 7 for the experimental scheme.

TABLE 6A Homeostasis model assessment of insulin resistance (HOMA-IR) EA B C D HOMA-IR (n = 3) (n = 11) (n = 10) (n = 6) (n = 2) Minimum 0.380.4363 0.3472 0.4363 0.6741 25% Percentile 0.38 0.5254 0.4223 0.67020.6741 Median 0.9086 0.958 0.5215 

1.472 0.8926 

75% Percentile 1.59 2.346 0.8414 2.054 1.111 Maximum 1.59 3.111 2.3362.77 1.111 Mean ± SEM 0.96 ± 0.35 1.35 ± 0.30 0.73 ± 0.19 1.45 ± 0.340.89 ± 0.0.22

TABLE 6B Homeostasis model assessment of beta cell function (HOMA % B).E A B C D HOMA % B (n = 3) (n = 11) (n = 10) (n = 6) (n = 2) Minimum 3813.96 20.73 22.8 38.57 25% Percentile 38 19.54 26.47 27.02 38.57 Median49.66 42.35 56.81 

57.27 

52.62 

75% Percentile 86.9 90 94.47 103.8 66.67 Maximum 86.9 112.5 225 110.866.67 Mean ± SEM 58.18 ± 14.75 50.97 ± 10.84 76.34 ± 21.44 63.01 ± 14.952.62 ± 14.05

FIG. 9 provides plots of circulating betatrophin levels for humansubjects. Sample size is indicated in parentheses. It is observed thatbetatrophin levels were increased at post-FMD refeeding compared to thebaseline and before administration of the FMD.

Table 7 shows that levels of adiponectin in the serum of human subjectson FMD were measured at baseline and after 3 cycles of the FMD,indicating a 40% increase in circulating adiponectin after 3 cycles ofthe diet (post-FMD, 7 days refeeding after cycle 3). Higher adiponectinlevels are associated with insulin sensitization and a lower risk oftype 2 diabetes.

TABLE 7 Levels of Adiponectin of human subjects before and afterreceiving 3 cycles of the FMD. pre-FMD post-FMD N 7 7 Mean 100 140.1 ±29.04

FIG. 10 provides charts of FMD cycles showing a reduction in cytokinesassociated with autoimmune type 1 diabetes in mice. MCP-1, TNFa, RANTESand IL-12 are known to be associated with the autoimmune pathogenesis oftype 1 diabetes. In the Streptozotocin (STZ)-induced T1D mouse model,levels of the indicated inflammatory cytokines in serum were reducedafter 8 cycles of FMD treatment.

Table 8 provides the effects of FMD on immune cells in mice. CytotoxicCD8+ T-cells have a major role in pathogenesis of type 1 diabetes.Numbers of cells (10⁶/ml) in the peripheral blood of STZ-induced T1Dmodel were measured at the 8^(th) cycle of the indicated timepoints,indicating a reduction of circulating T-cell populations in mice on FMDcycles. AL, ad libitum fed mice; FMD, at the end of FMD; post-FMD, 7days after post-FMD refeeding.

TABLE 8 Effects of FMD on immune cells in mice AL FMD post-FMD WBC 10.39± 1.1  6.81 ± 1.6 

9.23 ± 1.3    Total T cell  2.62 ± 0.34  1.34 ± 0.17 

2.18 ± 0.30 

CD8+ T cell 1.03 ± 0.2 0.58 ± 0.1 

0.34 ± 0.14 

While exemplary embodiments are described above, it is not intended thatthese embodiments describe all possible forms of the invention. Rather,the words used in the specification are words of description rather thanlimitation, and it is understood that various changes may be madewithout departing from the spirit and scope of the invention.Additionally, the features of various implementing embodiments may becombined to form further embodiments of the invention.

What is claimed is:
 1. A diet package comprising: rations for a fastingmimicking diet to be administered for a first time period to a subject,the fasting mimicking diet providing from 4.5 to 7 kilocalories perpound of subject for a first day and 3 to 5 kilocalories per pound ofsubject per day for a second to fifth day, the fasting mimicking dietbeing a low protein diet that provides less than 28 g of proteins on thefirst day and less than 18 g of proteins on the second to fifth days. 2.The diet package of claim 1 wherein the rations provide less than 30 gof sugar on the first day; less than 20 g of sugar on the second tofifth days.
 3. The diet package of claim 1 wherein the rations provide20 to 30 grams of monounsaturated fats on the first day.
 4. The dietpackage of claim 3 wherein the rations provide 10 to 15 grams ofmonounsaturated fats on the second to fifth days.
 5. The diet package ofclaim 1 wherein the rations provide between 6 and 10 grams ofpolyunsaturated fats on the first day and 3 to 5 grams ofpolyunsaturated fats on the second to fifth days.
 6. The diet package ofclaim 1 wherein the rations provide less than 12 g of saturated fats onthe first day and less than 6 grams of saturated fats on the second tofifth days.
 7. The diet package of claim 1 wherein the rations provide12 to 25 grams of glycerol per day on the second to fifth days.
 8. Thediet package of claim 1 wherein the rations provide over 5,000 IU ofvitamin A per day for days 1-5 and 60-240 mg of vitamin C per day fordays 1-5.
 9. The diet package of claim 8 wherein the rations provide400-800 mg of calcium per day for days 1-5; 7.2-14.4 mg of iron per dayfor days 1-5; 200-400 mg of magnesium per day for days 1-5; 1-2 mg ofcopper per day for days 1-5; 1-2 mg of manganese per day for days 1-5and 3.5-7 mcg of selenium per day for days 1-5.
 10. The diet package ofclaim 9 wherein the rations provide 2-4 mg of Vitamin B1 per day fordays 1-5; 2-4 mg of Vitamin B2 per day for days 1-5; 20-30 mg of VitaminB3 per day for days 1-5; 1-1.5 mg of Vitamin B5 per day for days 1-5;2-4 mg of Vitamin B6 per day for days 1-5; 240-480 mcg of Vitamin B9 perday for days 1-5; 600-1000 IU of Vitamin D per day for days 1-5; 14-30mg of Vitamin E per day for days 1-5; over 80 mcg of Vitamin K per dayfor days 1-5; and 16-25 mcg Vitamin B12 are provided during the entire5-day period.
 11. The diet package of claim 10 wherein the rationsprovide 600 mg of Docosahexaenoic acid (DHA, algae-derived) during theentire 5-day period.